Anti-Microbial Formulations Containing Nitric Oxide

ABSTRACT

Compositions and methods are directed to antimicrobial compositions and articles that release .NO from the composition and/or article in an amount effective to act as a microbicide. Preferred .NO-releasing compositions include a nitrosyl-containing organometallic compound, and can be encapsulated in gels or topical coatings.

This application is a continuation of and claims the benefit of U.S.patent application Ser. No. 13/168,120 filed on Jun. 24, 2011, which isa divisional of U.S. patent application Ser. No. 10/876,491, issued asU.S. Pat. No. 7,972,137 on Jul. 5, 2011, which claims the benefit ofU.S. Provisional Patent Application No. 60/483,236, which was filed Jun.30, 2003, and which are incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates generally to novel uses of nitric oxide (.NO) todeliver .NO for providing a microbicidal activity.

BACKGROUND OF THE INVENTION

With the discovery that the free radical .NO mimics the physiologicproperty of Endothelium-Derived Relaxing Factor (EDRF) (Furchgott andZawadzki, Nature, 288: 373-376, 1980; Palmer, et al., Nature, 327:524-526, 1987), it has become clear that .NO is an essential physiologicfactor for maintaining normal life processes. Among the importantphysiologic functions of .NO is its microbicidal activity when secretedfrom stimulated macrophages (see, e.g., S. H. Iyengar, et al., Proc.Natl. Acad. Sci. USA, 84: 6369-6373, 1987; Deng, et al., J. Immunol.151: 322-329, 1993; Weisz, et al., J. Biol. Chem. 269: 8324-8333, 1994;Fang, J. Clin. Invest. 99, 2818-2825; Shiloh, et al., Immunity, 10:29-38, 1999).

However, the pharmacological applications of .NO are limited, assystemic use can frequently result in severe toxicity. For instance,administration of .NO systemically to treat localized abnormalities ordiseases is contraindicated, because the control of its dosage in thetherapeutic range cannot be easily achieved. Even if it were possible tocarefully titrate the dose of .NO to minimize systemic toxicity, itwould be difficult to locally administer this gas to sites of interest.Therefore, the development of therapeutic agents, that mimic thepharmacological action of .NO, has received considerable attention.Several classes of .NO releasing compounds have been developed,including sydnonimine (Noack and Feelisch, J. Cardiovasc. Pharmacol.14S: 51-55, 1989), nitroglycerin (Noack and Feelisch, J. Cardiovasc.Pharmacol. 14S: 51-55, 1989), S-nitroso derivatives (Ignarro, et al., J.Pharmacol. Exp. Ther. 218: 739-729, 1981; Kowaluk and Fung, J.Pharmacol. Exp. Ther. 255: 1256-1254, 1990; Stamler, Loscalzo, Slivka,Simon, Brown and Drazen, U.S. Pat. No. 5,380,758, 1995) and N-nitrosocompounds (Maragos, et al., J. Med. Chem. 34: 3242-3247, 1991; Keefer,Dunams and Saavedra, U.S. Pat. No. 5,366,997; Keefer and Hrabie, U.S.Pat. No. 5,405,919). These compounds require either hydrolysis ormetabolic activation, through either oxidation or reduction, to generateNO.. In contrast nitroprusside, a member of a family of organic metalliccompounds, can release NO. through light activation (Wolf and Swinheart,Inorg. Chem. 14: 1049-1053, 1975; Bates, et al., Biochem. Pharmacol.42S: S157-S165, 1991).

Therefore, while the significance of .NO in numerous medicalapplications is well recognized and while there are numerous methods andcompositions known in the art that release .NO, all or almost all ofthem have various disadvantages. Consequently, there is still a need forcompositions and methods in which .NO can be safely and effectively usedin the treatment of a disease.

SUMMARY OF THE INVENTION

Embodiments of the inventive concept relate to compositions and methodsfor providing an antimicrobial activity to human tissue and compositionsfor providing such an activity. The methods and compositions provide acoating with a physiological acceptable polymer that encapsulates anitrosyl-containing organometallic compound, which can also be an ionicsalt or a chelate, that decomposes at body temperature and/or in thepresence of light to release .NO from the coating in an amount effectiveto have microbicidal activity.

One embodiment of the inventive concept is an antimicrobial compositionthat includes an .NO generating compound that is encapsulated in abiodegradable polymer, which is in turn formulated for use as a drugdelivery system. The .NO generating compound, for example anitrosyl-containing organometallic compound, decomposes to release anamount of .NO that is effective in providing an microbicidal activity.The composition can contain the nitrosyl-containing organometalliccompound, which can include a chelating agent, in amounts ranging from0.1 μmole to 100 μmoles. Suitable biodegradable polymers includegelatin, albumin, casein, fibrin, polylactide, polyglycocide,polyalkylene oxide, and polyvinyl chloride, and can be formulated toform a gel.

Another embodiment of the inventive concept is a method of providing anantimicrobial activity to human tissue by providing an .NO generatingcompound that is encapsulated in a biodegradable polymer, which is inturn formulated for use as a drug delivery system and applying it as acoating to the human tissue. Permitting the .NO generating compound (forexample, a nitrosyl-containing organometallic compound) to decompose toprovide .NO in amounts to be effective as a microbicide provides themicrobicidal activity. In some embodiments decomposition of the .NOgenerating compound is modulated by light. Such a nitrosyl-containingorganometallic compound can include a chelating agent, and can beprovided in amounts ranging from 0.1 μmole to 100 μmoles. Suitablebiodegradable polymers include gelatin, albumin, casein, fibrin,polylactide, polyglycocide, polyalkylene oxide, and polyvinyl chloride,and can be formulated to form a gel

Various objects, features, aspects and advantages of the presentinvention will become more apparent from the following detaileddescription of preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph depicting .NO concentration after exposure ofexemplary films to light.

FIG. 2 is a graph depicting microbicidal activity of .NO in an in vitroassay.

FIG. 3 is a perspective view of a dentist inserting a .NO releasing filminto a cavity in the gum of a patient.

DETAILED DESCRIPTION

The invention is based on the delivery of .NO from a coating in anamount effective to kill microbes that would otherwise result ingingival and other oral mucosal diseases. The coating includes anitrosyl-containing organometallic compound such that the compoundwithin the coating is stable at room temperature but at body temperatureand in the presence of light releases .NO from the coating. As usedherein, the term “nitrosyl-containing organometallic compound” includesionic salts and chelates. The method allows for the local delivery of.NO at concentrations at the surface of the gum that cannot be achievedsafely by the administration of a nitrosyl-containing organometalliccompound, including ionic salts or chelates, systemically, whether byapplying the compound itself, by intramuscular injection or by injectiondirectly into the blood vessel.

An example of such a nitrosyl-metal chelate is nitroprusside in the formof, for example, sodium nitroprusside. The iron ion of the nitroprussideis complexed to five cyano groups. The sixth ligand position of the ironion is occupied by a nitrosyl group. When a polymer having nitroprussideencapsulated therein is exposed to an aqueous solution and light, itdecomposes to release .NO (e.g., SNP in an aqueous solution uponexposure to light releases .NO, as detailed in Wolfe and Swinehart,Morgan. Chem. 14, 1049-1053, 1975). The enhanced stability ofnitroprusside in the polymer compared to its short lifetime in blood isthe result of the inability of compounds such as reductants, forexample, thiols and ascorbic acid, to interact with the nitroprussideby, for example, diffusing through small pores or the surface of thepolymer. Diffusion of these compounds into the polymer would inactivatethe nitroprusside by rapid decomposition and release of .NO. In somecases, however, it is desirable to include a reductant such as ascorbicacid or a thiol, to promote the release of .NO in the absence of light.

Other suitable complexing agents for the iron ion that can take theplace of the cyano groups of nitroprusside include, for example,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), and other members of this class of chelates;1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA),trans-1,2-cyclohexylenediamine-N,N,N′,N′-tetraacetic acid and othermembers of this class of chelates; as well as other chelates.

This invention relates to methods, compositions and articles ofmanufacture useful in the prevention of gingival disease. Byimpregnating a polymer with a nitrosyl-containing organometalliccompound, .NO can be locally delivered at a dose and at a rate that canbe controlled by varying, for example, the concentration of thenitrosyl-containing organometallic compound in the polymer, the natureof the polymer forming the coating, and the amount of time the polymerwith the nitrosyl-containing organometallic compound dispersed thereinis exposed to light. With such an approach, systemic .NO toxicity can beavoided.

Nitrosyl-containing organometallic compounds useful in the compositionare those which, when used according to the methods of the invention,are:

-   -   (a) non-toxic, that is, substantially free from any significant        toxic effects at their effective applied concentration;    -   (b) substantially free of symptomology, that is, they do not        produce significant symptoms detectable to the person treated at        their effective applied concentration; and    -   (c) relatively stable at room temperature in an aqueous        environment away from light, i.e., once a nitrosyl-metal chelate        is impregnated into a polymer, .NO is not released therefrom at        a significant rate, e.g., during the preparation of the coating        or during self storage in a packaged container, the        nitroprusside does not decompose at a rate greater than 0.1% per        month.

The nitrosyl-containing organometallic compound, can be a compound ofthe formula [MX5NO]-2 Y+2 or 2Y+1 where M is a transition metal such asFe, Co, Mn, Ru; X is a negatively a charged ion such as CN, Cl, Br, I,or one or more of the X ligands can be chelates such as EDTA, DTPA that,at physiologic pH, have negatively charged carboxylate groups; and Y isa positively charged ion, such as and alkali metal or alkaline earth,for example, Na+, K+ or similar monovalent cations or divalent cations.

Coatings according to the invention can be from 0.1-1.0 mm thick andcontain 1 μmole per mm² to 100 μmoles per mm² of the nitrosyl-containingorganometallic compound. Higher concentrations are desirable when thediffusion rate of .NO from the polymer is very slow or when it isdesirable to release higher concentrations of .NO, for example if themicroorganisms are resistant to the killing by low levels of .NO. Asused herein, “coatings” can refer to a coating on a substrate such as asheet, for example a plastic sheet or flexible sheet of thin metal; ormay refer to a sheet comprising a polymer formed on a surface having thenitrosyl-containing organometallic compound dissolved or dispersedtherein that is subsequently peeled away from the surface. In furthercontemplated aspects, the coating may also be directly applied to atooth or gingival tissue.

A wide variety of polymers can be used to encapsulate nitroprusside orother nitrosyl-containing organometallic compounds in forming thecoatings of the invention. Polymers include physiologically inertpolymers, biodegradable polymers, polymers that are only slowly soluble,and polymers that are insoluble in blood. Suitable insoluble polymersinclude those that form a permeable membrane coating so that .NO canmigrate from the coating as the .NO is produced. Suitable biodegradablepolymers that can be used as drug delivery systems include naturalpolymers, for example (1) collagen, (2) albumin, (3) casein, (4) fibrinand (5) gelatin (S. Bogdansky, in: Biodegradable Polymers as DrugDelivery Systems, ed. by M. Chasin and R. Langer, Marcel Dekker,. Inc.New York, pp. 231-259, 1990). Synthetic polymers suitable in forming thecoatings of the invention include: (1) polylactide and polyglycocide (D.H. Lewis, in: Biodegradable Polymers as Drug Delivery Systems, ed. by M.Chasin and R. Langer, Marcel Dekker,. Inc. New York, pp. 1-42, 1990);(2) polyvinyl alcohols (P. R. Byron and R. N. Dalby, J. Pharm. Sci. 76:65-67, 1987); and (3) polyalkylene oxides such as polyvinyl chloride.

Coatings of the invention can be applied to a foreign body, for examplea medical device that can be inserted or permanently implanted into aliving being, for example a human. When the foreign body is insertedinto the living being, it preferably is physiologically inert and, whenpermanently implanted, also biodegradable. Alternatively, the inventionexists as a polymer sheet that includes a nitrosyl-containingorganometallic compound dissolved or dispersed therein.

In use, it is contemplated that the coating (e.g., in form of a sheet orcoated foreign body) is placed in the mouth of a living being during adental procedure such as dental surgery. For example, the coating can beplaced against a gum or into a tooth socket after a tooth is extracted.Once in place, the coating is illuminated with light initiating therelease of .NO. The nitric oxide thus released acts as an anti-microbialto kill bacteria at and around the site where the coating is placed,effectively sterilizing the site where the procedure is being performed.Additional procedures or surgery can then continue. For example, after atooth socket has been sterilized, an implant can be placed in the toothsocket. This use thus allows for the procedure to proceed with gingivalor other oral mucosal disease being prevented or treated.

EXAMPLES Preparation of .NO Delivery System

A 5% (w/w) of polyvinyl alcohol (PVA, 99% hydrolyzed, M.W.124,000-186,000) solution was prepared by dissolving PVA in distilledwater at 100° C. After this PVA solution was prepared, it was cooled toroom temperature and nitroprusside, as sodium nitroprusside (SNP), wasadded to reach a final concentration of 10 mM. A solution of polyvinylchloride (PVC) (prepared by dissolving PVC in THF, 5% w/w) was placedover a glass plate and air-dried. This dried PVC film was coated witheither PVA-containing SNP or PVA-without SNP and allowed to air dry inthe dark. Then a solution of PVC (5%, w/w in THF) was again placed overthe dried PVA-containing SNP/PVC film. In this manner, SNP, which wasincorporated into PVA, is sandwiched between two layers of PVC, creatinga thin film. At this point, these two-types of films, PVC/PVA-containingSNP and PVC/PVA without SNP were used to estimate .NO release andmeasure microbicidal activity.

Assay for .NO

The Griess Reaction was used to estimate .NO concentration afterexposure of the two types of films prepared according to Example 1, i.e.PVC/PVA-containing SNP and PVC/PVA without SNP, to light. For theseexperiments 1-cm×1-cm squares of each film (n=3) were cut, placed into asolution of 2 mL of pH 7.4 sodium phosphate buffer, and exposed to lightfrom a slide projector for various times. After which the films wereremoved and the remaining solution was exposed to air for 24 hr. Asample of the phosphate buffer (0.6 mL) was removed and added to thefreshly prepared Griess reagent (0.4 mL of 0.1% N-(1-naphthyl)ethylenediamine in water and 1% sulfanilamide in 5% phosphoric acidmixed 1:1). This reaction was incubated for 15 min at room temperatureand absorbance was recorded at 550 nm. Concentrations of nitrite wereestimated by comparing absorbances at 550 nm against standard solutionsof sodium nitrite prepared in the same buffer (Green, et al., Anal.Biochem. 126: 131-138, 1982) Data are presented in FIG. 1.

Microbicidal Activity Assay of .NO

Experiments were undertaken to evaluate the microbicidal properties of.NO released from sodium nitroprusside incorporated into a PVC/PVA film,which is then exposed to light. In Phase 1, preliminary studies wereperformed in order to determine the appropriate testing procedures forlight activated .NO release and its antimicrobial properties. Theselected test organism was Pseudomonas aeruginosa (ATCC 15542). Thisselection was based on its usefulness in AOAC protocols to determinedisinfection and effects of antimicrobial agents. Knowing thatPseudomonas aeruginosa grows in a slime layer, allowed for evaluation ofthe usefulness of the PVC/PVA film containing sodium nitroprusside andthe ability of .NO released into a semi-liquid medium, as might be foundin the oral cavity. In addition, various sources of light wereevaluated. A high intensity dental light, which is often used in thecuring of composite materials in the dentist's office was initiallyevaluated. In addition, standard incandescent bulb light with a shieldto focus the light to a localized area was also evaluated. Allexperiments were performed in duplicate by overlapping procedures sothat the procedure was repeated at least one time in addition to addingother techniques. In Phase 2, time, medium, organism and conditions ofexposure were selected from the preliminary trials and evaluatedregarding antimicrobial effects of released .NO from the PVC/PVA film.

In a typical phase 2 experiment, MH plates (100×15 mm, 8 cm in diameter)were divided in half and labeled side A and side C. Plates wereinoculated with 500 ml of the microbial suspension (˜1.0×105 CFU/ml) andspread plated to cover the agar plate. An additional plate was spreadplated and incubated undisturbed (control). Pre-cut sections of film Aand film C were placed on their appropriate side. The negative (nolight) control was placed under a box to restrict light exposure. All ofthe test plates were labeled individually for 3 or 5 min (representingthe time of exposure to the light). Post-exposure, all plates wereincubated at 37° C. for 24 hr., the PVC film removed, the clear zone, ifany, evaluated and placed back in the incubator. The evaluation wasrepeated at 48 hr.

All plates exposed to .NO released from sodium nitroprussideincorporated into a PVC/PVA film, demonstrated a clear outlined squarewhere the film had been placed and extending beyond. By 48 hr thetreatment zones extended to include 100% of the plates. The control filmand inoculum control showed the characteristic light green color usuallyassociated with Pseudomonas aeruginosa and its growth on Mueller Hintonagar. Results are graphically depicted in FIG. 2. These data at 24 hrand 48 hr were interpreted to indicate microbial killing associated withthe released .NO from sodium nitroprusside incorporated into a PVC/PVAfilm.

In FIG. 3, a dentist 10 is inserting a .NO releasing film 20 into acavity 30 in the gum of a patient 40. To the side is a packaging 50 fromwhich the film was removed, the packaging having text 52 instructing useof the film for dental use. The term “dentist” is used here as aeuphemism to indicate any person performing dental work, includingsomeone having a dental degree and license, a veterinary degree if thepatient is an animal, a licensed or non-licensed assistant, dentalstudent, and so forth. The term “dental use” should be similarly broadlyconstrued.

In general, the dentist would produce a cavity 30 in the gum of apatient, such as by extraction of a tooth, or by removal of other tissuesuch as occurs in a root canal operation. The term “film” here is usedto mean any relatively thin piece, especially one not more than 5 mm inthickness. Of course it may be that the dentist did produce the cavityhim/herself. The cavity may have been produced as a natural course of adisease process, through a traumatic accident, by an assistant, or insome other manner.

It is possible that the film would be marketed in small squares or othershapes, and the dentist would simply select one or more pieces from acontainer. Alternatively, the dentist could cut a larger piece of film20 into a suitable smaller size. Scissors could be used to sever thefilm into an appropriately sized piece, or the film could be segmented,torn, or severed in some other manner.

The next step is to introduce the film into the cavity 30. This could beaccomplished in any suitable manner, introducing the film as a thinsheet, or as a folded or crumpled sheet, or indeed as a pellet. At thispoint there are two distinct procedures that could be followed. In oneprocedure the dentist waits until it is expected that a suitable amountof .NO has been released from the film, removes the film from thecavity, and closes the cavity. Alternatively, the dentist could insert adissolving form of film into the cavity, and then suture or otherwiseclose the cavity with the film inside. In either case it is contemplatedthat the film would release sufficient .NO to effectively treat amicroorganism promoted gingival disease by significantly reducing theprevalence of active microorganisms. In some instances the dentist maychoose to shine a light onto the film, which light would trigger rapidrelease of .NO from the film. If a light is not used, release of the .NOcould be triggered by enzymes or other components naturally present inthe mouth.

The film can utilize any suitable .NO releasing material, but preferablyuses a nitrosyl-containing organometallic compound, which mayadvantageously be present as an ionic salt or a chelate. The mostpreferred .NO releasing material is a nitroprusside salt such as sodiumnitroprusside.

Thus, specific embodiments and applications of anti-microbial dentalformulations for the prevention and treatment of oral mucosal diseasehave been disclosed. It should be apparent, however, to those skilled inthe art that many more modifications besides those already described arepossible without departing from the inventive concepts herein. Theinventive subject matter, therefore, is not to be restricted except inthe spirit of the appended claims. Moreover, in interpreting both thespecification and the claims, all terms should be interpreted in thebroadest possible manner consistent with the context. In particular, theterms “comprises” and “comprising” should be interpreted as referring toelements, components, or steps in a non-exclusive manner, indicatingthat the referenced elements, components, or steps may be present, orutilized, or combined with other elements, components, or steps that arenot expressly referenced.

What is claimed is:
 1. An antimicrobial composition comprising: an .NOgenerating compound encapsulated within a biodegradable polymerformulated for use as a drug delivery system, wherein the .NO generatingcompound decomposes to release .NO in an amount effective to havemicrobicidal activity.
 2. The antimicrobial composition of claim 1,wherein the biodegradable polymer is formulated to form a gel.
 3. Theantimicrobial composition of claim 1, wherein the antimicrobialcomposition comprises a coating, and wherein the coating is configuredto be directly applied to human tissue.
 4. The antimicrobial compositionof claim 1, wherein the .NO generating compound is a nitrosyl-containingorganometallic compound.
 5. The antimicrobial composition of claim 4,wherein the nitrosyl-containing organometallic compound is present at0.1 μmole to 100 μmoles.
 6. The antimicrobial composition of claim 4,wherein the nitrosyl-containing organometallic compound comprises achelating agent.
 7. The antimicrobial composition of claim 1, whereinthe biodegradable polymer includes at least one member of the groupconsisting of gelatin, albumin, casein, fibrin, polylactide,polyglycocide, polyalkylene oxide, and polyvinyl chloride.
 8. Theantimicrobial composition of claim 1, wherein the .NO generatingcompound decomposes to generate an amount of .NO effective as abactericide.
 9. A method of providing antimicrobial activity to humantissue comprising: providing an .NO generating compound encapsulatedwithin a biodegradable polymer formulated for use as a drug deliverysystem; applying the encapsulated .NO generating compound encapsulatedwithin the biodegradable polymer as a coating to a human tissue; andallowing the .NO generating compound to decompose to generate an amountof .NO effective as a microbicide.
 10. The method of claim 9, whereinthe biodegradable polymer is formulated to form a gel.
 11. The method ofclaim 9, wherein the .NO generating compound is a nitrosyl-containingorganometallic compound.
 12. The method of claim 11, wherein thenitrosyl-containing organometallic compound is present at 0.1 μmole to100 μmoles.
 13. The method of claim 11, wherein the nitrosyl-containingorganometallic compound comprises a chelating agent.
 14. The method ofclaim 9, wherein the biodegradable polymer includes at least one memberof the group consisting of gelatin, albumin, casein, fibrin,polylactide, polyglycocide, polyalkylene oxide, and polyvinyl chloride.15. The method of claim 9, wherein the decomposition of the .NOgenerating compound is modulated by exposure to light.
 16. The method ofclaim 9, wherein the .NO generating compound decomposes to generate anamount of .NO effective as a bactericide.